title: Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli.
abstract: The SAP6 gene (without signal sequence) encoding Metschnikowia reukaufii acid protease was amplified by PCR and fused to the expression vector pET-24a(+). The carboxy-terminal 6x His-tagged recombinant acid protease (rSAP6) was expressed from pET-24a(+)SAP6-6His in Escherichia coli BL21 (DE3) and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified rSAP6 was 54kDa. The optimal temperature and pH of the purified rSAP6 were 40 degrees C and 3.4, respectively. The enzyme was stable below 45 degrees C and between pH 2.6 and 5.0. The results show that Mn(2+) had an activating effect on the enzyme, while Cu(2+), Mg(2+), Zn(2+) and Ag(+) acted as inhibitors of the enzyme. However, Ca(2+) had no effect on the enzyme activity. The purified rSAP6 was characterized as an aspartic protease as it was inhibited by aspartic protease-specific inhibitors, such as pepstatin. It was also found that the purified rSAP6 had milk-clotting activity.
authors.affiliation: UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, Shandong 266003, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Zhenming
authors.initials: Z
authors.lastname: Chi
authors.forename: Xianghong
authors.initials: X
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D055163:Metschnikowia / Q000201:enzymology* / Q000235:genetics; D008892:Milk / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010436:Pepstatins / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008670:Metals; D010436:Pepstatins; D011993:Recombinant Fusion Proteins; D057055:Aspartic Acid Proteases; C031375:pepstatin